SUMMARY Sustained release of drugs delivered onto mucosal surfaces by oral, topical or inhaled routes can be a highly effective means of improving topical bioavailability. Prolongation of epithelial residence time is especially relevant to anti-infective therapies in diseases characterized by chronic infection and inflammation where treatment needs to be both locally potent and systemically safe. Inhaled antibiotics have been used to treat chronic airway infections and have transformed the management of cystic fibrosis (CF), an inherited disease of thickened, abnormally viscoelastic and poorly transported airway mucus leading to persistent lung infection, which affects approximately 30,000 patients in the US and 80,000 world-wide. Despite these benefits, dose- limiting and potentially toxic serum concentrations are an inherent risk of bolus inhalation delivery, especially for drugs with systemic bioavailability. In order to address this we have developed a novel therapeutic solution based on conjugation of inhaled drugs to a natural airway protein modified to have strong mucus-binding properties. C35STrx, a monocysteinic active-site variant of thioredoxin-1 (Trx) remains bound covalently to soluble and tethered airway mucus for many hours, and does not induce inflammation, irritation, or adverse reactions. In preliminary studies the C35STrx scaffold could be conjugated to over ten individual linkers (each of which can be attached modularly to one drug molecule) without loss of target-binding activity. With typical mucus clearance times of 20-24 hours such mucus-bound C35STrx-drug conjugates are anticipated to persist on the airway surface and allow controlled release of drug payloads over a much longer duration and at lower maximum systemic exposure levels than possible with inhaled delivery of free drug molecules. This phase I application is focused on proof of concept using the potent antibiotic vancomycin as a payload. Specifically, in Aim 1) we will construct C35STrx-vancomycin conjugates using modular cleavable linkers and characterize their physical properties, size, cleavability and stability; in Aim 2) we will evaluate the conjugates in vitro for valency (drug:scaffold ratio), mucus protein binding activity and antibacterial activity (minimum inhibitory concentration) of cleaved vancomycin conjugates vs. free, non-conjugated vancomycin; and in Aim 3) we will determine the lung inflammation status, in vivo pharmacokinetics and airway residence time following intratracheal (IT) delivery to mice of vancomycin-C35STrx conjugates in both the reduced (target binding) and oxidized (inert) forms as compared to free vancomycin lacking the C35STrx scaffold. The pharmacokinetics of IT-delivered vancomycin conjugates will be compared to that of free vancomycin delivered intravenously. If the Aims of this project are achieved key validation of the feasibility of sustained mucosal release of active drug payloads conjugated to C35STrx will be established. This will enable subsequent Phase II activities to further develop and optimize sustained delivery of vancomycin or other antibiotics leading to human trials of potentially safer and more efficacious inhalation treatment regimens for CF.